A Single Intrathecal or Intraperitoneal Injection of CB2 Receptor Agonist Attenuates Bone Cancer Pain and Induces a Time-Dependent Modification of GRK2

The objective of this study was to explore the potential role of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cannabinoid 2 receptor (CB2) agonist-induced analgesic effects of bone cancer pain. Female Sprague–Dawley rats, weighing 160–180 g, were utilized to establish a model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells. JWH-015, a selective CB2 agonist, was injected intrathecally or intraperitoneally on postoperative day 10. Bone cancer-induced pain behaviors—mechanical allodynia and ambulatory pain—were assessed on postoperative days ?1 (baseline), 4, 7, and 10 and at post-treatment hours 2, 6, 24, 48, and 72. The expressions of spinal CB2 and GRK2 protein were detected by Western Blotting on postoperative days ?1 (baseline), 4, 7, and 10 and at post-treatment hours 6, 24, and 72. The procedure produced prolonged mechanical allodynia, ambulatory pain, and different changes in spinal CB2 and GRK2 expression levels. Intrathecal or intraperitoneal administration of JWH-015 alleviated the induced mechanical allodynia and ambulatory pain, and inhibited the downregulation of spinal GRK2 expression. These effects were in a time-dependent manner and reversed by pretreatment of CB2 selective antagonist AM630. The results affirmed CB2 receptor agonists might serve as new treatment targets for bone cancer pain. Moreover, spinal GRK2 was an important regulator of CB2 receptor agonist-analgesia pathway.     

Bacterial tail anchors can target to the mitochondrial outer membrane

Background

During the generation and evolution of the eukaryotic cell, a proteobacterial endosymbiont was re-fashioned into the mitochondrion, an organelle that appears to have been present in the ancestor of all present-day eukaryotes. Mitochondria harbor proteomes derived from coding information located both inside and outside the organelle, and the rate-limiting step toward the formation of eukaryotic cells may have been development of an import apparatus allowing protein entry to mitochondria. Currently, a widely conserved translocon allows proteins to pass from the cytosol into mitochondria, but how proteins encoded outside of mitochondria were first directed to these organelles at the dawn of eukaryogenesis is not clear. Because several proteins targeted by a carboxyl-terminal tail anchor (TA) appear to have the ability to insert spontaneously into the mitochondrial outer membrane (OM), it is possible that self-inserting, tail-anchored polypeptides obtained from bacteria might have formed the first gate allowing proteins to access mitochondria from the cytosol.

Results

Here, we tested whether bacterial TAs are capable of targeting to mitochondria. In a survey of proteins encoded by the proteobacterium Escherichia coli, predicted TA sequences were directed to specific subcellular locations within the yeast Saccharomyces cerevisiae. Importantly, TAs obtained from DUF883 family members ElaB and YqjD were abundantly localized to and inserted at the mitochondrial OM.

Conclusions

Our results support the notion that eukaryotic cells are able to utilize membrane-targeting signals present in bacterial proteins obtained by lateral gene transfer, and our findings make plausible a model in which mitochondrial protein translocation was first driven by tail-anchored proteins.

Reviewers

This article was reviewed by Michael Ryan and Thomas Simmen.

     

Bond length pattern associated with charge carriers in armchair graphene nanoribbons

The geometry configuration of charged armchair graphene nanoribbons (AGNRs) is theoretically investigated in the framework of a two-dimensional tight-binding model that includes lattice relaxation. Our findings show that the charge distribution and, consequently, the bond length pattern is dependent on the parity of the nanoribbon width. In this sense, the lattice distortions decrease smoothly for increasingly wider GNRs. As should be expected, AGNRs belonging to a particular family present similar patterns for the bond lengths. The interplay between the electron-phonon coupling and band gap is also investigated. The results show that the electron-phonon coupling strength is fundamental to promote the transition from metallic towards semiconducting-like behavior for the band gap. Most important, such strength is crucial on defining the degree of lattice distortions in AGNRs.     

Functional roles of T3.37 and S5.46 in the activation mechanism of the dopamine D1 receptor

The activation mechanism of dopamine receptors is unknown. The amino acids S5.42, S5.43, and S5.46 located in helix 5 appear to be crucial, but their specific roles in receptor activation have not been studied. We modeled the D1 dopamine receptor using the crystal structures of the D3 dopamine and β2 adrenergic receptors. Molecular dynamics simulations show that the interaction of dopamine with the D1 receptor leads to the formation of a hydrogen-bond network with its catechol group and helices 3, 5, and 6, including water molecules. The para hydroxyl group of dopamine binds directly to S5.42 and N6.55, the latter also interacting with S5.43. Unexpectedly, S5.46 does not interact directly with the catechol; instead, it interacts through a water molecule with S5.42 and directly with T3.37. The formation of this hydrogen-bond network, part of which was previously observed in docking studies with dopamine agonists, triggers the opening of the E6.30–R3.60 ionic lock associated with the activation of GPCRs. These changes do not occur in the unbonded (apo) receptor or when it is in a complex with the antagonist 3-methoxy-5,6,7,8,9,14-hexahydrodibenz[d,g]azecine. Our results provide valuable insight into the T3.37–S5.46–water–S5.43–ligand interaction, which may be crucial to the activation of the D1 dopamine receptor and should be considered during the design of novel agonists.

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Graphical Abstract General representation of the relationship between the formation of the HBN and the opening of the R3.50–E6.30 ionic lock

     

Efficient isolation method for high‐quality genomic DNA from cicada exuviae

In recent years, animal ethics issues have led researchers to explore nondestructive methods to access materials for genetic studies. Cicada exuviae are among those materials because they are cast skins that individuals left after molt and are easily collected. In this study, we aim to identify the most efficient extraction method to obtain high quantity and quality of DNA from cicada exuviae. We compared relative DNA yield and purity of six extraction protocols, including both manual protocols and available commercial kits, extracting from four different exoskeleton parts. Furthermore, amplification and sequencing of genomic DNA were evaluated in terms of availability of sequencing sequence at the expected genomic size. Both the choice of protocol and exuvia part significantly affected DNA yield and purity. Only samples that were extracted using the PowerSoil DNA Isolation kit generated gel bands of expected size as well as successful sequencing results. The failed attempts to extract DNA using other protocols could be partially explained by a low DNA yield from cicada exuviae and partly by contamination with humic acids that exist in the soil where cicada nymphs reside before emergence, as shown by spectroscopic measurements. Genomic DNA extracted from cicada exuviae could provide valuable information for species identification, allowing the investigation of genetic diversity across consecutive broods, or spatiotemporal variation among various populations. Consequently, we hope to provide a simple method to acquire pure genomic DNA applicable for multiple research purposes.     

Pregnancy Rates in Repeat-breeder Heifers Following Multiple Artificial Inseminations during Spontaneous Oestrus

Hormonal asynchronies during oestrus, related to the presence of suprabasal plasma-progesterone (P4) concentrations and a delayed ovulation, interfere with the fertility of repeat-breeder heifers (RBH). Since tubal dysfunction can occur in connection with hormonal asynchronies and constrained availability of fertile spermatozoa at the time of ovulation, the present study tested the hypothesis that frequent sperm deposition from onset of oestrus to ovulation may improve pregnancy rates in RBH. Five RBH and five virgin heifers (VH; controls) were repeatedly artificially inseminated (AI) at 6 h intervals from onset of oestrus to spontaneous ovulation. Hormone analyses revealed suprabasal P₄ concentrations and a delay in the occurrence of the luteinising hormone (LH) surge, but a normal cortisol profile in RBH. Compared with controls, RBH presented longer interval from onset of oestrus to ovulation, and therefore, received more AIs. Pregnancy rates in RBH reached control levels (60%; NS), indicating that the hypothesis might be correct. Pregnancy rates in VH were below the expected range, presumably attributed to a deleterious influence of the frequent handling. The study suggests that pregnancy rates can be improved in RBH by frequent AI in relation to spontaneous ovulation. However, this practice of repeated manipulations, while seeming not to show adverse effects, lacks practicality for routine use.     

16S rRNA Phylogeny of Sponge-Associated Cyanobacteria

Phylogenetic analyses of 16S rRNA sequences of sponge-associated cyanobacteria showed them to be polyphyletic, implying that they derived from multiple independent symbiotic events. Most of the symbiont sequences were affiliated to a group of Synechococcus and Prochlorococcus species. However, other symbionts were related to different groups, such as the Oscillatoriales.     

Adiponectin is associated with bone mineral density in perimenopausal women.

The aim of the current investigation was to investigate any potential effect of fasting plasma adiponectin concentration on bone tissue, and to find possible relationships of fasting plasma adiponectin level with different body composition, insulin sensitivity and physical performance parameters in a group of healthy perimenopausal women. Twenty-one premenopausal and 17 early postmenopausal women participated in this study. The women were matched for body mass index (BMI) and level of mean daily energy expenditure. Women had similar adiponectin (8.4 +/- 3.9 vs. 9.9 +/- 5.4 microg/ml) and leptin values (12.0 +/- 7.7 vs. 14.0 +/- 8.2 ng/ml) before and after menopause. Significant relationships were observed between plasma adiponectin and bone mineral content, total bone mineral density (BMD) and lumbar spine BMD values (r > - 0.36; p < 0.05). Furthermore, adiponectin had a significant negative association with total BMD (beta = - 1.228; p = 0.004) and lumbar spine BMD (beta = - 0.312; p = 0.005) independent of the influence that other measured body compositional, hormonal or physical performance factors may exert on BMD. Adiponectin was also significantly related to waist-to-hip ratio (WHR) (beta = - 2.300; p = 0.002) and fasting insulin resistance index (FIRI) (beta = - 0.006; p = 0.007) in separate regression models. No relationship was observed between leptin and measured bone, physical performance and insulin resistance values. Leptin significantly correlated to BMI (beta = 0.018; p = 0.034), lean body mass (beta = 0.025; p = 0.024) and fat mass (beta = 0.019; p = 0.001) in separate regression models. In conclusion, the results of present study show that circulating adiponectin appears to exert an independent effect on BMD in perimenopausal women and may represent a link between adipose tissue and bone mineral density.     

The impact of target site accessibility on the design of effective siRNAs

Small-interfering RNAs (siRNAs) assemble into RISC, the RNA-induced silencing complex, which cleaves complementary mRNAs. Despite their fluctuating efficacy, siRNAs are widely used to assess gene function. Although this limitation could be ascribed, in part, to variations in the assembly and activation of RISC, downstream events in the RNA interference (RNAi) pathway, such as target site accessibility, have so far not been investigated extensively. In this study we present a comprehensive analysis of target RNA structure effects on RNAi by computing the accessibility of the target site for interaction with the siRNA. Based on our observations, we developed a novel siRNA design tool, RNAxs, by combining known siRNA functionality criteria with target site accessibility. We calibrated our method on two data sets comprising 573 siRNAs for 38 genes, and tested it on an independent set of 360 siRNAs targeting four additional genes. Overall, RNAxs proves to be a robust siRNA selection tool that substantially improves the prediction of highly efficient siRNAs.